This invention is in the field of angiogenesis inhibitors, in particular antibiotics that inhibit angiogenesis.
Angiogenesis, the proliferation and migration of endothelial cells that result in the formation of new blood vessels, is an essential event in a wide variety of normal and pathological processes. For example, angiogenesis plays a critical role in embryogenesis, wound healing, psoriasis, diabetic retinopathy, and tumor formation, as reported by Folkman, J. Angiogenesis and its inhibitors. In: V. T. DeVita, S. Hellman and S. A. Rosenberg (eds.). Important Advances in Oncology, pp. 42-62, (J. B. Lippincott Co., Philadelphia, 1985); Brem, H., et al., Brain tumor angiogenesis. In: P. L. Kornblith and M. D. Walker (eds.), Advances in Neuro-Oncology, pp. 89-101. (Future Publishing Co., Mount Kisco, N.Y. 1988); Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med., 285; 1182-1186 (1971); and Folkman, J. Successful treatment of an angiogenic disease. N. Engl. J. Med., 320: 1211-1212 (1989).
Identification of several agents that inhibit tumor angiogenesis has provided a conceptual framework for the understanding of angiogenesis in general. The inhibition of angiogenesis by certain steroids and heparin derivatives, reported by Folkman, J., et al., Science 221: 719. (1983); and Murray, J. B., et al., Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor. J. Biol. Chem., 261: 4154-4159 (1986); led to studies elucidating the crucial role of remodeling of the extracellular matrix in angiogenesis. These agents apparently prevent angiogenesis by specifically disrupting the deposition and cross-linking of collagen, as reported by Ingber, D., and Folkman, J. Inhibition of angiogenesis through modulation of collagen metabolism. Lab. Invest., 59: 44-51 (1989).
The original description of angiogenesis inhibition in the presence of cartilage, reported by Brem, H., et al., J. Exp. Med. 141: 427-439 (1975); Brem, H., et al., Extracellular Matrices Influences on Gene Expression pp. 767-772 (Academic Press, NY 1975); and Langer, R., et al., Science 70-72 (1976); led to the isolation and purification from bovine cartilage of a protein fraction that not only inhibited angiogenesis but inhibited protease activity, described by Murray, J. B., et al., J. Biol. Chem. 261: 4154-4159 (1986). Subsequently, an extract derived from the vitreous of rabbits was shown to inhibit tumor angiogenesis by Brem, S., et al., Am. J. Ophthal. 84: 323-328 (1977). The demonstration that heparin alone enhanced the angiogenesis response buttressed the hypothesis that heparin produced by mast cells that had migrated to the tumor site facilitated the development of new capillaries, as reported by Kessler, D. A., et al., Int. J. Cancer 18:703-709 (1976).
Recent studies on inhibition of angiogenesis have highlighted the importance of enzyme mediated remodeling of the extracellular matrix in capillary growth and proliferation (Folkman, J., et al., Science 221: 719-725 (1983),; Ingber, D., et al. Lab. Invest. 59: 44-51 (1989); Folkman, J., et al., Science 243: 1490-1493 (1989); Krum, R., et al., Science 230: 1375-1378 (1985); Ingber, D., et al., Endocrinol. 119: 1768-1775 (1986); and Ingber, D., et al., J. Cell. Biol. 109: 317-330 (1989)). It has been suggested Ingber, D., et al., Lab. Invest. (1989) and Endocrinol. (1986) that the steroid-heparin combination is involved in the dissolution of the capillary basement membrane by inhibiting the deposition or cross-linking of collagen. The isolation of a collagenase inhibitor from cartilage, the first source of an angiogenesis inhibitor, was of interest since it suggested that angiogenesis may be inhibited not only by disrupting collagen deposition, but also by interrupting collagen breakdown. It is therefore possible that a combination of agents that interfere with both the anabolic and catabolic phases of collagen metabolism will prove even more effective in halting tumor angiogenesis.
A number of investigators have reported that extracts of cartilage, one of the few avascular tissues in the body, can inhibit angiogenesis: Eisentein, et al., Am. J. Pathol. 81, 1-9 (1987); Pauli, et al., J. Natl. Cancer Inst. 67,55-74 (1981); Brem and Folkman, J. Exp. Med. 141, 427-439 (1975); Langer, et al., Science 193, 70-72 (1976); Langer, et al., Proc. Natl. Acad. Sci. USA 77, 431-435 (1980); and Lee and Langer, Science 221, 1185-1187 (1983). Langer, et al., showed that cartilage extracts containing a collagenase inhibitor retard tumor-induced and inflammatory-induced neovascularization in the cornea and conjunctiva, when delivered by either infusion or sustained release from a polymeric implant.
Although extracts from several different tissue sources have been shown to contain anti-angiogenic activity, as reviewed by D""Amore, Prop. Clin. Biol. Res. 221, 269-283 (1986), and several molecules have been found which inhibit different aspects of angiogenesis, such as cell proliferation or cell migration, no single tissue-derived macromolecule capable of inhibiting angiogenesis has been identified in the prior art. However, other inhibitors of collagenase are known that do not inhibit angiogenesis, including xcex12-macroglobulin and tissue inhibitor or metalloproteinase (TIMP). TIMP inhibits capillary endothelial cell proliferation but not angiogenesis. Protamine inhibits blood vessel ingrowth but not proliferation of capillary endothelial cells.
The potential therapeutic benefit that an effective, economic, well characterized inhibitor of angiogenesis might have in controlling diseases in which neovascularization plays a critical role has prompted a long term search for angiogenesis inhibitors. There are many advantages to having such an inhibitor that can be prepared by simple organic synthesis, rather than by cloning and expression of a protein, or synthesis of a long polypeptide. An inhibitor of angiogenesis could have an important therapeutic role in relieving the course of these disorders, as well as provide a valuable means of studying their etiology.
It is therefore an object of the present invention to provide a pharmaceutical composition, and method of use thereof, for the treatment of diseases involving abnormal angiogenesis.
It is another object of the present invention to provide topical and controlled release pharmaceutical compositions, and methods of use thereof, for inhibition of angiogenesis.
It is still another object of the present invention to provide an economical, well characterized, composition for inhibition of angiogenesis.
An antibiotic composition that is an effective inhibitor of angiogenesis has been developed. The preferred antibiotic is minocycline, although other tetracycline-like antibiotics that inhibit collagenase are also effective.
The effective dosage for inhibition of angiogenesis in vivo is extrapolated from in vitro and in vivo inhibition assays. Effective dosages range from approximately one micromolar to 500 micromolar. The dosage range is much higher than the dosage used for inhibition of bacterial growth. The effective dosage is somewhat dependent on the method and means of delivery. For example, in some applications, as in the treatment of psoriasis or diabetic retinopathy, the inhibitor is delivered in a topical carrier. In other applications, as in the treatment of solid tumors, the inhibitor can be delivered by means of a biocompatible, biodegradable or non-degradable, polymeric implant, systemically, or by local infusion.
Effectiveness was demonstrated by incorporating minocycline into controlled release polymers and testing in the rabbit cornea against neovascularization in the presence of the VX2 carcinoma. Inhibition by minocycline was shown to be comparable to that of the combination of heparin and cortisone, a potent inhibitor of angiogenesis. Minocycline decreased tumor-induced angiogenesis by a factor of 4.5, 4.4 and 2.9 at 7, 14 and 21 days, respectively. At the end of the study, none of the corneas with minocycline had such vascular masses, in contrast to the corneas with empty polymers, which had large, invasive, exophytic tumors. Further studies demonstrate the selectivity of the compound for endothelial cells.